The present invention relates to the use of genetically-modified strains of Drosophila melanogaster in high-throughput screening (HTS) of small molecular weight compounds. The present invention also relates to the systematic identification of small molecules which interfere with specific disease pathways using Drosophila as the genetic model system. Additionally, the present invention relates to the use of automated screening systems employing microinjection of small molecular weight compounds, possessing putative biological activity, into the open circulatory system (i.e., the hemolymph) of genetically-modified Drosophila larva.
The identification and subsequent characterization of new therapeutics are the primary rate limiting step in pharmacological research. Drug discovery processes are extremely lengthy. A conventional process involves the screening of thousands of individual compounds for a desired biological/therapeutic activity. Historically, less than 1 in 10,000 of the synthetic compounds have actually been approved by the Food and Drug Administration (FDA), at a cost of greater than $200 million per drug (see e.g., Ganellin, et al., 1992. In: Medicinal Chemistry for the 21st Century. pp. 3-12 (Blackwell Publications, London, England)).
Pharmacological compounds have been sought from natural products for many years. In general, complex mixtures derived from cells, or their secondary metabolites, are screened for biological activity. Subsequently, when the desired biological activity is identified in such a complex mixture, the specific molecule which possesses the activity has been purified, using the biological activity as the means for identifying the component of the mixture which possesses the desired biological activity.
An alternative methodology for the development of novel pharmacological compounds has been to screen individual compounds which have been previously synthesized and saved in xe2x80x9clibrariesxe2x80x9d within drug/chemical companies or research institutions. The compounds in these libraries were often initially chosen for synthesis or screening due to the fact that they possessed a particular functionality thought to be relevant to a specific biological activity.
More recently, peptide or oligonucleotide libraries have been developed which may be screened for a specific biological function (see e.g., Musser, 1992. In: Medicinal Chemistry for the 21st Century. pp. 3-12 (Blackwell Publications, London, England); U.S. Pat. Nos. 5,593,853 and 5,639,603). For example, recombinant peptide libraries have been generated by the insertion of degenerate oligonucleotides into the genes encoding the capsid proteins of filamentous bacteriophage and the DNA-binding protein Lac I (see e.g., Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87:6378-6382; Cull, et al., 1992. Proc. Natl. Acad. Sci. U.S.A. 89:1865-1869 and PCT Publications Nos. WO 91/19818 and WO 93/08278). These random libraries may contain more than 109 different peptides, each fused to a larger protein sequence which is physically-linked to the genetic sequences encoding it. The libraries are subsequently screened by allowing the peptide to interact with its specific ligand (e.g., receptor, nucleic acid, etc.) via several rounds of affinity purification and the selected exposition or display vectors are then amplified in, for example, E. coli, and the nucleic acid contained therein is sequenced to reveal the identity of the peptide responsible for the ligand interaction.
Many of the existing therapeutics on the market to date have been identified in an accidental manner, and frequently their mechanism of action is poorly understood. A more direct approach towards the identification of new small molecular weight compounds effective against various disease conditions requires the precise knowledge of both the molecular defect underlying a given disease and the knowledge of the cellular pathways and processes in which the defective component is acting. In fact, such knowledge of the pathways involved is essential since the defective gene product may not be the best target for a small molecular weight compound. In addition, despite the great value that large libraries of molecules can have for identifying useful compounds or improving the properties of a lead compound, the difficulties of screening such libraries, particularly extremely large libraries, has limited the impact access to such libraries should have made in reducing the costs of drug discovery and development. This is, in a large part, due to the weaknesses inherent in the current screening methodologies of compound libraries which employ both cell-free and in vitro cell-based assay systems.
Currently, numerous drug screening protocols rely upon high-throughput screening (HTS) of compound libraries using cell-free or in vitro cell-based assay systems. Several drugs (e.g., cyclosporine A and mevastatin) have emerged directly from utilization of this methodology. HTS is a process by which large numbers of compounds with putative biological activity may be tested, preferably in an automated manner, for activity as inhibitors (antagonists) or activators (agonists) of a specific biological target (e.g., cell-surface receptor or a metabolic enzyme). It should be noted, however, that HTS does not actually identify a drug, but rather, the primary goal of HTS is to identify high-quality xe2x80x9chitsxe2x80x9d or xe2x80x9cleadsxe2x80x9d (i.e., compounds which affect the target molecule in the desired manner) which are active at a relatively low concentration and that possess a novel structure or sequence and to supply directions for their potential optimization. Although HTS is a powerful screening tool, it possesses a number of limitations such as: (i) bioavailability; (ii) pharmacokinetics; (iii) toxicity and (iv) absolute specificity. Hence, subsequent medicinal chemical and pharmacological studies are required to convert a compound which emerges from an initial HTS screening into a therapeutically useful drug. These limitations exist because many of the properties critical to the development of a drug typically can not be directly assessed by HTS; therefore, the final compound which eventually becomes a drug is unlikely to have been the molecule present in the initial small molecular weight compound library. Generally, the greater the number and diversity of the compounds which are analyzed, the more successful the screening is likely to be, a fact which has markedly accelerated the development of HTS.
A well-designed HTS screening assay may also provide information regarding the potency of a compound of interest. Generally, the lower the concentration at which the compound of interest exhibits activity, the more likely it will exhibit specificity and, as a corollary, the less likely that it will have undesirable or deleterious side-effects. Information on specificity may also be obtained by concomitantly performing a counter-screen with a related target molecule (e.g., an HIV protease verses a cellular aspartyl protease or the serotonin 2A receptor verses the serotonin 2C receptor). Compounds which exhibit activity only against the primary target are deemed most likely to possess the necessary selectivity. If different chemotypes may be identified using the same screen, then medicinal chemists will have a broader range of options for modification of the novel, lead compound. In addition, the spectrum of compounds which score positive (and to some extent those compounds which score negative) may help to pinpoint those structural characteristics and motifs of the molecules which are responsible for their efficacy and specificity.
The HTS methodology requires four distinct elements: (i) suitably arrayed compound libraries; (ii) as assay methodology amenable to automation; (iii) a robotics workstation and (iv) a computerized system for input and analysis of incoming data from the screening assay. Currently, the 96-well microtiter plate is the standard format for automated HTS assay, although arrays of compounds on chips (see e.g., Fodor, et al., 1993. Nature 364:555-556) or on insoluble beads (see e.g., Ohlmeyer, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90:10922-10926) have also been utilized and the assays may be performed on agar plates or other types of solid support matrices. The synthesis of combinatorial libraries may be accomplished within the 96-well microtiter plates, thereby providing addresses for the particular compounds generated by a given subset or series of reactions and thus identifying the compound of interest. Furthermore, concentrates of fermentation broths, natural product extracts or pre-existing collections of compounds (i.e., such as the repositories possessed by large pharmaceutical firms) may be dispensed in 96-well microtiter plates, either singly (simplex arrays) or as defined mixtures of 10-20 compounds per well (multiplex arrays). The later, multiplex array methodology permits a far more rapid rate of screening, but it also requires the subsequent deconvolution of the compounds within the assay mixture to identify the true active compound.
Robotics systems for HTS assays range in complexity from simple, automated dilution devices to highly evolved workstations in which multiple functions are performed by one or more mechanical arms. In the preferred embodiment of the HTS methodology, fall automation (i.e., from sample dispensing to data collection) allows for round-the-clock operation, thereby increasing the overall screening rate and mitigating the potential for human error common in highly redundant procedures such as HTS. Given the variety of libraries which are currently available, the large number of compounds present in each library and the need to compare the results obtained from different screening assays, data collection and management are critical to automated HTS. Databases of structural characteristics, assays performed, screening results and the like, must be relational (i.e., interlinked) so as to allow the necessary information to be extracted by a query from any perspective. Hence, one should be able to search, for example, for all compounds which are active at a certain threshold concentration in a particular screen, or for the characteristics of all compounds of similar structure in different screens.
Although any standard drug activity assay may, in theory, be performed utilizing a HTS methodology, the conversion to a fully automated process imposes certain, frequently formidable, constraints which affect the design of the assay in practice. Procedures which are routine when performed on the bench (e.g., centrifugation to remove cellular debris or to collect the beads, rinsing of the wells in the microtiter plate during an ELISA-based assay and the like) are often extremely difficult to automate. The greater the number of steps required in the assay, the greater the difficulty in developing automation. The ideal HTS assay is one in which all required manipulations may be performed in a single well of the microtiter plate.
Current screening methodologies of compound libraries employ both cell-free and in vitro cell-based assay systems, although each of these aforementioned systems possesses intrinsic limitations and weaknesses. For example, in the case of the cell-free systems, screening is limited to single candidate target molecules. Such putative targets are generally identified upon the basis of enzymatic activity (e.g., kinases) or specific protein domains involved in protein-protein interaction (e.g., src-homology 2 (SH2) domains) (see e.g., Broach, et al., 1996. Nature 384:14-16). Another potential limitation of the cell-free assay systems is that they do not provide an inherent test for either the specificity of the interaction, nor of the toxicity of the particular compound. In addition, the prime drug target for a given disease condition is ideally the weakest point within the signaling chain. Cell-free systems do not permit the identification of such targets, as signaling pathways are made up of combination of cell membrane, cytoplasmic, and nuclear-based components whose complex interactions are disrupted in cell-free systems.
In contrast, cell-based assays have several notable advantages over cell-free systems. First, the starting material (the cell) is self-replicating, thus avoiding the investment involved in the preparation of a purified target, in chemically-modifying the target to suit the specific type of screening assay, and the like. Second, the targets and readouts are examined in a biological context which (hopefully) mimics the normal physiological conditions present in vivo. Third, cell-based assay systems can provide insights regarding bioavailability (i.e., the compound must enter the cell to affect an intracellular target) and cytotoxicity (whether a compound compromises cellular processes and growth). However, while in vitro cell-based assays more closely model a compound""s specificity, toxicity or possible mode of action within a cell, they often provide inadequate similarity to the in vivo disease condition, as most diseases develop within multicellular tissues. The results in a cell-based system are limited to such criteria as changes in the mitotic rate or reporter gene expression. Nonetheless, cell-based systems are becoming more frequently utilized as an alternative to in vitro biochemical assays for HTS.
Generally, such in vitro cell-based assays require the ability to examine a specific cellular process and a means to measure its output. For example, agonist-mediated activation of a cell-surface receptor or a ligand-gated ion channel may be followed by monitoring its coupled cellular response (see e.g., Levitzki, 1996. Curr. Opin. Cell Biol. 8:239-244). Although the immediate downstream event (e.g., transient elevation in intracellular CA2+ levels, phosphorylation of target proteins and the like) may be difficult to evaluate in a quantitative automated format, the subsequent transcriptional changes may be more amenable to such monitoring. For example, binding of isoproterenol to the xcex2-adrenergic receptor elicits a transient rise in cyclic AMP (cAMP) levels, activating protein kinase A (PKA), which translocates to the nucleus and phosphorylates a transcription factor (CREB) which subsequently recognize cAMP response elements (CREs). Accordingly, CREB activation may be detected and quantified by measuring the expression level of a reporter gene whose transcription is driven by an enhancer element containing CREs (see e.g., Kee, et al., 1996. J. Biol. Chem. 271:2373-2375). Enhancer elements which couple gene expression to distinct signal transduction pathways have now been characterized (see e.g., Treisman, 1996. Curr. Opin. Cell Biol. 8:205-215) and reporter genes which generate products that can be readily adapted to the HTS format are also currently available and include, but are not limited to: xcex2-galactosidase, luciferase, alkaline phosphatase, xcex2-lactamase and the green fluorescent protein Oellyfish).
The fruitfly, Drosophila melanogaster, has been used as an ubiquitous model for the characterization of cellular processes (e.g., signaling pathways) involved in a variety of human diseases. In fact, the cellular functions of many genes known to be affected in human diseases were initially identified in Drosophila (see e.g., Holley, et al., 1997. Bioassays 19: 218-284). This high degree of conservation of morphogenetic processes between Drosophila and humans has made Drosophila a prime model system for the identification of putative drug targets using function based genetic approaches. Homeotic genes constitute one of the best-known examples of genes first identified in Drosophila that have provided insight into the mechanisms of human development and disease (see e.g., van Heyningen, 1997. Mol. Med. 3:231-237).
Drosophila eye development is a prime example of a model system for the study of well defined and functionally integrated genetic controls. Drosophila eye development has provided most of the important genetic information regarding evolutionarily conserved mechanisms. One of the earliest transcriptional regulatory regions controlling Drosophila eye development, the PAX6 gene, was initially defined in humans and the mouse as, respectively, the gene mutated in aniridia (absence of the iris; see e.g., Ton, et al., 1991. Cell 67:1059-1074) and in Small Eye strains (see e.g., Hill, et al., 1991. Nature 354:522-525). Subsequently, it was discovered that a Drosophila homologue existed that mapped to the eyeless locus, and that the homolog was functionally disrupted by transposon insertion in eyeless fruitflies (see e.g., Quiring, et al., 1994. Science 265:785-789). Another novel finding involving carefully controlled ectopic expression of either the fly PAX6 gene or the mouse homologue in different Drosophila imaginal disks was shown to lead to the development of relatively xe2x80x9cnormalxe2x80x9d eyes and antennae (see e.g., Halder, et al., 1995. Science 267:1788-1792). This high degree of conservation of both gene sequence and function, across a broad phylogenetic spectrum, has served as a major incentive for a reaffirmation of the concept of wide-ranging evolutionary gene conservation (see e.g., Banfi, et al., 1996. Nat. Genet. 13:167-174).
While the concept of wide-ranging evolutionary gene conservation is not new, it has been critical in the elucidation of many complexities in, for example, the Ras and tyrosine kinase signaling pathways via the analysis of Drosophila eye mutants. These genes encode components of a highly conserved signaling cascade which has in part been described in vertebrate cells. Activation of the sev (sevenless) receptor by boss (bride of sevenless) presumably results in receptor dimerization and subsequent autophosphorylation on tyrosine residues. This autophosphorylation creates binding sites for the Drk SH2/ SH3 adaptor protein (see e.g., Raabe, et al., 1995. EMBO J 14:2509-2518). Drk binds to sev via its SH2 domain and to the C-terminus of Sos via its SH3 domains and thereby brings the Sos protein to the membrane. Sos is a guanine nucleotide releasing factor that activates Ras1 by facilitating the conversion from GDP-Ras1 to GTP-Ras1. Ras1 in turn activates a cascade of three cytoplasmic kinases: homologs of Raf, MEK (MAPK kinase) and MAP kinase encoded by the genes raf, Dsor1, and rolled, respectively (Dickson, et al., 1996 Genetics 142:163-171). All of these cytoplasmic components are also required for signaling by other RTKs in Drosophila, including both torso and DER (see e.g., Chang, et al., 1994. Cold Spring Harbor Symp. Quant. Biol. 59:219-226; Dominguez and Hafen, 1996. Drosophila Sem. Cell Dev. Biol. 7:219-226). The elucidation of these signaling pathways are of great importance as they control various aspects of human developmental regulation, hormone action, and neoplasia.
Genetically manipulated Drosophila have served as a powerful tool for dissecting and characterizing gene pathways. Recombinant methodologies used to modify specific Drosophila genes are well known in the art, as are methodologies used for the maintenance of said Drosophila strains, said strains being homozygous, hemizygous, or heterozygous for defined allelic combinations of the gene or genes of interest. One such phenotype is an irregular, rough eye surface induced upon activation of a genetically modified raf gene in the Ras signaling pathway. In such a genetically sensitized background, mutations in genes coding for rate limiting components in a particular signaling pathway can be identified as modifiers of the phenotype (see e.g., Dickson, et al., 1996. Genetics 142:163-171). These mutations thus identify gene products whose activity or function is critical for the development the disease-related phenotype.
Extensive genetic screens for mutations in genes involved in the transduction of the signal from the activated receptors to the nucleus have revealed an evolutionarily conserved signaling cascade that is used by the different receptors to elicit diverse cellular responses. The components of the signaling pathways between Drosophila and humans are highly conserved. Inhibitors developed against human proteins also block the function of Drosophila proteins. For example, the immunosuppressant drug rapamycin has been shown to block the activation of ribosomal protein S6 kinase (S6K) in mammalian and Drosophila cells.
The Drosophila compound eye has been used extensively for the systematic genetic dissection of conserved signaling pathways. Its structure and usefulness in characterization of signaling pathways is therefore discussed in some detail. The Drosophila compound eye is composed of a hexagonal array of approximately 800 identical units, called ommatidia. Each of these units consists of eight photoreceptor cells (R1-R8), four lens-secreting cone cells, and pigment cells that optically insulate each ommatidium. This highly organized structure develops from a single layer epithelial sheet, the eye imaginal disc, during larval and pupal stages by the stepwise recruitment of cells into the ommatidial clusters. During this process, differentiating cells specify the fate of neighboring, but still undetermined, cells by inductive signals.
As previously discussed, a variety of cell-fate decisions are controlled by the activation of receptor tyrosine kinases (RTKs). One such interaction involves the Drosophila rough eye phenotype which is an irregular, rough eye surface induced upon activation of a genetically-modified raf gene (a downstream effector of the Ras1 oncogene) in the Ras signaling pathway (see e.g., Dickson, et al., 1996. Genetics 142:163-171). In such a genetically-sensitized background, mutations in genes encoding rate limiting components in a particular signaling pathway may be identified as modifiers of the phenotype. These mutations thus identify gene products whose activity or function is critical for the development the disease-related phenotype.
Another function of RTK involves the control of R7 photoreceptor cells during the development of the compound eye of Drosophila. The inductive signal specifying the fate of the R7 cell is well-understood since mutations in two genes, sevenless (sev) and bride of sevenless (boss), specifically block the specification of the R7 precursor as a photoreceptor cell giving rise to ommatidia which contain seven instead of eight photoreceptors. The boss gene encodes a membrane protein that is exclusively expressed on the surface of the R8 cell at the time when the presumptive R7 photoreceptor cell is recruited into the cluster. The sev gene encodes a receptor tyrosine kinase expressed in a subpopulation of cells within the ommatidial clusters including the R7 precursor and the four cone-cell precursors. The boss protein binds to and activates the sev receptor on the neighboring R7 precursor and, in the absence of either boss or sev function, the R7 precursor fails to initiate neuronal differentiation and instead develops as a non-neuronal cone cell. Expression of constitutively activated forms of sev or the ubiquitous expression of the boss gene showed that sev activation is sufficient to specify the R7 fate not only in the R7 precursor cell but also in the four cone-cell precursors. As illustrated in FIG. 1, during the development of each ommatidial unit, five cells, collectively referred to as the R7 equivalence group, are competent to choose between two alternative fates: the neuronal photoreceptor fate when sev is active and the non-neuronal cone cell fate when sev is inactive.
The formation of R7 photoreceptor cells is a model system where scientists have a complete knowledge of all genes acting in the signal transmission pathway that extends from the cell membrane to the nucleus (see e.g., Dominguez, et al., 1997. Dev. Biol. 7:219-226). Although extensive genetic screens for recessive, viable mutations affecting the development of the R7 cells have been carried out, mutations in only four genes apart from sev and boss have been identified. Flies homozygous for mutations in the sina gene (which encodes a nuclear protein) lack R7 cells and are non-viable. Mutations in Gap1 (encoding a homolog of a GTPase activating protein), yan (encoding a transcription factor of the ETS-family) and tramtrack (ttk) have been shown to cause the formation of multiple R7 cells in each ommatidium (see e.g., Dominguez, et al., 1997. Dev. Biol. 7:219-226). These results tends to suggest that Gap1, yan and ttk act as inhibitors of the R7 specification; whereas sina acts as an activator.
Mutations in genes whose products are also involved in other developmental decisions prior to the formation of the eye may cause less informative phenotypes such as lethality so that their role in R7 development cannot be tested directly and alternative genetic strategies are thus required to identify them. The most successful approach to date has been pioneered by Simon et al. (1991, Cell 67:701-716) and focuses on the development of the R7 cell. In this study, researchers utilized a hypomorphic sev mutation which encoded a partially functional, temperature-sensitive sev receptor which, at an intermediate temperature, provides barely sufficient activity to specify R7 cells. In this background, a 50% reduction in the amounts of an essential, rate-limiting component due to the inactivation of one gene copy by a mutation was demonstrated to prevent the formation of R7 cells. In this sensitized genetic background, normally recessive mutations thus acted dominantly in the R7 decision. A similar, genetically-sensitized system was also provided by the multiple R7 phenotype in sev gain of function (GOF) mutations (sevGOF). In this assay, the overall number of cells of the R7 equivalence group which assume R7 cell fate provide a sensitive measure of the sev kinase activity and, additionally, of the efficiency with which the signal is transduced. The recruitment of extra R7 cells disrupts the hexagonal array of the ommatidial units and causes a roughening of the external surface of the eye. Mutations that reduce the efficiency of sev signaling are thus detected as suppressors of the rough eye phenotype of sevGOF27. It is by the utilization of such simple and reliable F1 screens that many of the genes for cytoplasmic signaling components involved in sev signal transduction have been discovered.
The value of Drosophila as a screening system for evaluating the biological activities of chemicals has been well-documented (see e.g., Schulz, et al., 1955. Cancer Res. 3(suppl.): 86-100; Schuler, et al., 1982. Terat. Carcin. Mutag. 2:293-301). Small numbers of chemical substances are administered to larvae or flies by feeding, and flies are then analyzed for survival and for phenotypic alterations. Although these conventional tests show the potential use of Drosophila as a tool to analyze the function of small molecular weight compounds, these methods neither permit high-throughput screens, nor permit the directed search for small molecular weight compounds that interfere with a specific morphogenetic pathway related to a human disease condition. Application of compounds by feeding requires relatively large amounts of the substance, and its uptake by the larvae and thus its final concentration is, at best, difficult to control. Furthermore, application by feeding does not permit automation of the procedure necessary for high-throughput analysis.
The principal property required of a small molecular weight compound screen is that distinct mechanisms of induction produce different outcomes. Response patterns can thus be used to group drugs with similar mechanisms and hence identify novel activities. To be successful, such patterns have to be relatively insensitive to potency, so that agents with the same mechanism but different potency are classed together. The principle to be exploited is that the differences in the panel cells"" capacities to respond depend on the components and assembly of their signal transduction and effector mechanisms for differentiation. With the current screens using cell-free or in vitro cell-based assays, these differentiation end-points are difficult to assess and cell number or cell mass may be the more appropriate assay for their high-throughput designs. This is due to the fact that current screening methodologies can not easily discriminate growth arrest due to differentiation from other antiproliferative or simple cytotoxic effects (see e.g., Francis, et al., 1994. Differentiation 57:63-75).
Whole embryo cultures have also been used to screen for chemical effects in, for example, rodents and chickens. Adverse embryonic outcomes (malformations or embryotoxicity) are directly related to the serum concentration of the compound being tested. These serum concentrations can be directly compared to the serum concentration in the human. Whole embryo culture systems are problematic in that they result in large numbers of in vivo false-positives, and development within the cultures is limited to the very early stages of embryogenesis (see e.g., Webster, et al., 1997. Int. J. Dev. Biol. 41:329-335). Similarly, the nematode Caenorhabditis elegans is frequently utilized as a model organism for the genetic dissection of developmental controls and cell signaling. However, in C. elegans there are no genetically sensitized systems available that permit reliable detection of even a two-fold reduction in a signaling process caused by either a chemical compound or a mutation in a gene. Although C. elegans can be grown in microtiter plates, the phenotypic screens are markedly limited. Also, chemical compounds would necessarily be administered by feeding, and would thus possess all of the aforementioned inherent disadvantages.
Another widely-utilized model genetic system is yeast. Although yeast are easily maintained and can readily be grown in large numbers, they are a simple, single-celled organism and thus possess the inherent limitation of being incapable of replicating a complex, multi-cellular system. Although the yeast system offers a comparatively higher throughput, its possesses inherent limitations, as most disease conditions are dependent upon cell-cell interactions within tissues that cannot be modeled in yeast. Finally, and most importantly, the overall degree of conservation of signaling pathways between yeast and human is significantly lower than that between Drosophila and humans.
Accordingly, there remains an as yet unfulfilled need within the relevant fields for a rapid, quantitative HTS screening methodology which has the ability to be fully automated and that utilizes a genetic model possessing, but not be limited to, the following characteristics: (i) a high degree of conservation of the various signaling pathways involved in the etiology of human disease; (ii) the ability to be grown rapidly in large numbers with little effort; (iii) a stable genetic mutation(s) and (iv) an easily discernible genetic outcome for use in the screening procedure.
The present invention targets the ubiquitous nature of the signaling pathways present in most every cell and couples them to an assay system of choice within the appropriately modified Drosophila strain. More specifically, the present invention utilizes genetically sensitized Drosophila strains which possess mutations within a selected signaling pathway in the construction of a high-throughput screen (HTS) of small molecular weight compounds to facilitate the identification and characterization of novel, lead drug candidates which dominantly modify the phenotypic effects of these aforementioned sensitized Drosophila signaling pathways.
The present invention discloses a methodology for the screening of compounds for desirable biological/therapeutic activities which involves the screening of individual chemical compounds which have been synthesized and cataloged in libraries of drug or chemical companies or research institutes. The active xe2x80x9cleadxe2x80x9d compounds and novel chemical entities identified and characterized by the present invention may be utilized for the development of bioactive xe2x80x9cleadsxe2x80x9d in small molecule libraries for pharmaceuticals, agrochemicals and the like.
The high degree of conservation of morphogenetic processes between Drosophila and humans has made Drosophila a prime model system for the identification of new putative drug targets using function-based genetic approaches. Genetically sensitized Drosophila systems, wherein the gene modification results in a dose-sensitive phenotype, permit detection of a mere two-fold effect of small compounds on specific signaling pathways related to human diseases. It is preferred that such Drosophila strains are genotypic (+/null), and hence are hemizygous for a dose sensitive gene within a given signaling pathway.
The present invention is a novel combination of an automated, in vitro HTS assay with an in vivo xe2x80x9creadoutxe2x80x9d system comprised of Drosophila melanogaster which are genetically-sensitized for a specific disease pathway, preferably a human disease pathway. Hence, the present invention discloses a methodology which serves to xe2x80x9cbridge the gapxe2x80x9d of the presently-utilized screening systems by use of a well-established model for human disease (Drosophila) for the screening of large numbers of compounds for biological/therapeutic activity in a rapid, quantitative and highly efficacious manner. The expression of human disease genes or their homologs within the developing Drosophila larva models the effects of these genes in human cells and subsequently produces the phenotypes which are modified by either the mutations within these interacting genes, or by compounds which block the function of the corresponding gene product. These gene products are prime candidates as targets for small compounds which interfere with their function.
The preferred embodiment of the present invention employs an automated system for the microinjection of compound(s) of interest into the open circulatory system (i.e., hemolymph) of Drosophila larvae, most preferably of Drosophila third instar larvae, that have been previously genetically-modified in a gene specific to a signaling pathway involved in human disease. More preferably the modified gene is involved in the Ras signaling pathway and has a functional phenotype that is readily scorable by observation of the developing Drosophila eye. However, it is contemplated that the invention can use any signaling pathway to monitor phenotypic changes involved in the development of any imaginal disk. Following maturation of the microinjected Drosophila larvae, the biological effect of the injected compound(s) are assessed in adult flies. In addition, the screening methodology disclosed by the present invention allows the simultaneous observation of: (i) the general biological toxicity of the microinjected compound(s) through 50% lethal dose (LD50) computations: (ii) the specificity of the modification of the specific Drosophila phenotype (i.e., suppression of the rough eye phenotype) and (iii) the non-specific interference with other well-defined developmental and physiological pathways.